The invention relates to methods for sterilizing materials and preparing vaccines.
Various methods and devices exist for the sterilization, decontamination, or disinfection of biological and non-biological materials. These methods include thermal destruction (e.g., burning), heat sterilization, irradiation (e.g., ultraviolet or ionizing irradiation), gas sterilization (e.g., using ethylene oxide), photosensitization, membrane sterilization, or the use of chemical disinfectants (formaldehyde, glutaraldehyde, alcohols, mercury compounds, quaternary ammonium compounds, halogenated compounds, solvent/detergent systems, or peroxides).
Heat sterilization (e.g., autoclaving) is often used, for example, for sterilizing medical solutions prior to use in a patient. Heat sterilization typically requires heating a solution to 121xc2x0 C. for a minimum of 15 minutes under pressure in an autoclave, maintaining the heat and pressure conditions for a period of time sufficient to kill bacteria, fungi, and protists and inactivate viruses in the solution.
Many reusable medical articles and materials are not suitable for disinfection or sterilization in an autoclave. For example, plastic parts on medical devices, hemodialyzers, and fiber optic devices are commonly sterilized by chemical germicide treatment. In general, germicides require several hours of treatment for the inactivation of microorganisms.
To ensure sterility in pharmaceutical production, gas sterilization is often employed. However, gas sterilization (e.g., using ethylene oxide) can be time-consuming, requiring prehumidification, heating, and evacuation of a sample chamber, followed by treatment with high concentrations of the gas for up to 20 hours at a time. When properly used, traditional disinfectants can inactivate vegetative bacteria, certain fungi, and lipophilic or medium-sized viruses. However, these disinfectants often do not arrest tubercle bacillus, spore-forming bacteria, or non-lipophilic or small-sized viruses.
Another method for lysing cells, and thereby sterilizing a sample is described in Microbiology (Davis et al., Harper and Row, Hagerstown, Md., 1980). This procedure of freezing and thawing the sample is believed to exert its effect through formation of tiny pockets of ice within the cells when a suspension of bacteria is frozen. The ice crystals and the high localized concentrations of salts both cause damage to the bacteria. A single freezing event is generally sufficient to kill only some of the bacteria, but repeated freeze-thaw cycles result in a progressive decrease in viability. Lethality is correlated with slow freezing and rapid thawing.
Traditional freeze-thaw methods are limited in the speed of the freeze-thaw cycle by the time needed to transfer heat to and from the center of the sample to effect phase changes. The equilibrium rate is particularly slow in the case of large volume samples (e.g., about 100 ml or larger). Sterilization efficiency of the traditional methods is limited by the impracticality of performing a large number of freeze-thaw cycles by those methods.
Traditional methods of food preservation include pasteurization, in which a food is held at an elevated temperature for a period of time.
There is presently a need to develop methods for inactivating microbes and viruses from protein preparations while maintaining the integrity and therapeutic value of the proteins. The development of methods for inactivation of non-encapsulated viruses is especially challenging, since the outer coats of such viruses generally include proteins similar to the proteins one wishes to retain.
The invention is based on the discovery that biological and non-biological materials can be sterilized, decontaminated, or disinfected by repeatedly cycling between relatively high and low pressures. Pressure cycling can be carried out at low, ambient, or elevated temperatures (e.g., from about xe2x88x9240xc2x0 C. to about 95xc2x0 C., e.g., xe2x88x9240xc2x0 C., xe2x88x9235xc2x0 C., xe2x88x9230xc2x0 C., xe2x88x9225xc2x0 C., xe2x88x9220xc2x0 C., xe2x88x9215xc2x0 C., xe2x88x9210xc2x0C., xe2x88x925xc2x0 C., 0xc2x0 C., 4xc2x0 C., 5xc2x0 C., 10xc2x0 C., 15xc2x0 C., 20xc2x0 C., 25xc2x0 C., 30xc2x0 C., 35xc2x0 C., 37xc2x0 C., 40xc2x0 C., 45xc2x0 C., 50xc2x0 C., 55xc2x0 C., 60xc2x0 C., 65xc2x0 C., 70xc2x0 C., 75xc2x0 C., 80xc2x0 C., 85xc2x0 C., 90xc2x0 C., 95xc2x0 C., or intermediate ranges). New methods based on this discovery can have applications in, for example, the preparation of vaccines, the sterilization of blood plasma or serum, plant, animal, and human tissue, sputum, urine, feces, water, and ascites, the decontamination of military devices, food and beverage production, and the disinfection of medical equipment. The new methods can also be incorporated into production processes or research procedures.
In general, in one embodiment, the invention features a method for sterilizing a material that includes at least one desired macromolecule (e.g., a nucleic acid, a protein, a lipid, a carbohydrate, a drug, a steroid, or a nutrient). The method includes the steps of providing the material at an initial pressure; and increasing the pressure to an elevated pressure sufficient to sterilize the material but insufficient to irreversibly inactivate the biological activity of the desired macromolecule.
The invention also features a method for sterilizing a material initially contaminated with at least one infectious agent selected from the following: a bacterium, a prion, a virus, an infectious nucleic acid, or an infectious protein. The method includes the steps of providing the material at an initial temperature and pressure; and increasing the pressure to an elevated pressure sufficient to sterilize the material. The initial temperature is generally lower than 60xc2x0 C.
Examples of contaminants that can be destroyed or inactivated by these new methods include, but are not limited to, bacteria, prions, viruses, fungi, protists, nucleic acids, and proteins.
In some cases, the method can also include decreasing the pressure to a decreased pressure, and cycling the pressure between the decreased pressure and the elevated pressure at least two times (e.g., 2, 3, 4, 5, 6, 8, 10, 20, 25, 50, 100, 250, 500, 1000 times or more). The decreased pressure can be the same as or different from the initial pressure, and is typically (although not necessarily) about half of the elevated pressure or less. Thus, if the elevated pressure is 40,000 psi, the decreased pressure will generally be 20,000 psi, 10,000 psi, 5,000 psi, 1,000 psi, 500 psi, 250 psi, 100 psi, 50 psi, 20 psi, 1 atm or less, or any intermediate value.
The invention also features a method for sterilizing a material. The method includes the steps of providing the material at an initial temperature and pressure; increasing the pressure to an elevated pressure sufficient to sterilize the material; decreasing the pressure to a decreased pressure; and repeating the increasing and decreasing steps at least once. In this method, the initial temperature can be, for example, about 40xc2x0 C. or lower.
The material sterilized by the above methods can be, for example, a biological sample; blood plasma, serum, or other plant, animal (including insects, mammals, reptile, etc.), or human tissue; feces; urine; sputum; medical or military equipment; a foodstuff; a pharmaceutical preparation; ascites; a vaccine; or any other material to be sterilized.
The initial pressure can be, for example, atmospheric pressure (i.e., about 1 atm, or about 14.7 psi), or a lower pressure (less than 1 atm, e.g., 0.01 psi, 0.1 psi, 1 psi, 10 psi, or intermediate pressures) or a higher pressure (greater than 1 atm, e.g., 20 psi, 50 psi, 100 psi, 200 psi, 500 psi, 1000 psi, 2000 psi, 5000, 10000 psi, 20,000 psi, or higher). The material can be provided at an initial temperature in the range of from about xe2x88x9240xc2x0 C. to about 95xc2x0 C. (e.g., xe2x88x9240xc2x0 C., 35xc2x0 C., xe2x88x9230xc2x0 C., xe2x88x9225xc2x0 C., xe2x88x9220xc2x0 C., xe2x88x9215xc2x0 C., xe2x88x9210xc2x0 C., xe2x88x925xc2x0 C., 0xc2x0 C., 4xc2x0 C., 5xc2x0 C., 10xc2x0 C., 15xc2x0 C., 20xc2x0 C., 25xc2x0 C., 30xc2x0 C., 35xc2x0 C., 37xc2x0 C., 40xc2x0 C., 45xc2x0 C., 50xc2x0 C., 55xc2x0 C., 60xc2x0 C., 65xc2x0 C., 70xc2x0 C., 75xc2x0 C., 80xc2x0 C., 85xc2x0 C., 90xc2x0 C., 95xc2x0 C., or intermediate ranges), less than xe2x88x9240xc2x0 C. or lower (e.g., xe2x88x9240xc2x0 C., xe2x88x9250xc2x0 C., xe2x88x9260xc2x0 C., xe2x88x9270xc2x0 C., xe2x88x9280xc2x0 C. or lower), or 95xc2x0 C. or higher (e.g, 95xc2x0 C., 100xc2x0 C., or higher).
The elevated pressure can, for example, be in the range of about 5,000 psi to about 120,000 psi (e.g., 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000 psi, or intermediate ranges), although lower pressures such as 100 psi, 500 psi, 1000 psi, or 2000 psi can be useful in some applications.
Optionally, the new methods can also include warming or cooling the material prior to or after the pressure-increasing step.
The invention also features a method for sterilizing a material, the method including the steps of providing a material at an initial pressure (e.g., 1 atm) and temperature (e.g., xe2x88x9240xc2x0 C., xe2x88x9235xc2x0 C., xe2x88x9230xc2x0 C., xe2x88x9225xc2x0 C., xe2x88x9220xc2x0 C., xe2x88x9215xc2x0 C., xe2x88x9210xc2x0 C., xe2x88x925xc2x0 C., 0xc2x0 C., 4xc2x0 C., 5xc2x0 C., 10xc2x0 C., 15xc2x0 C., 20xc2x0 C., 25xc2x0 C., 30xc2x0 C., 35xc2x0 C., 37xc2x0 C., 40xc2x0 C., 45xc2x0 C., 50xc2x0 C., 55xc2x0 C., 60xc2x0 C., 65xc2x0 C., 70xc2x0 C., 75xc2x0 C., 80xc2x0 C., 85xc2x0 C., 90xc2x0 C., 95xc2x0 C., or intermediate ranges); increasing the pressure to an elevated pressure insufficient to irreversibly denature proteins (i.e., less than about 50,000 psi), but still high enough to kill at least some (e.g., at least 25%, 50%, 75%, 90%, 95%, 99%, or even substantially all) pathogens that contaminate the material (e.g., in the range of about 5,000 psi to about 120,000 psi, e.g., 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000 psi, or intermediate ranges); and subsequently decreasing the pressure to the initial pressure or thereabouts, to provide a sterilized material.
The material can be chilled to a subzero temperature (e.g., from about xe2x88x9240xc2x0 C. to about 0xc2x0 C., especially between about xe2x88x9220xc2x0 C. and about xe2x88x925xc2x0 C.) either before or after the pressure is increased. The temperature can be subsequently increased, either before or after the pressure is decreased.
The pressure can optionally be repeatedly cycled (e.g., 2, 3, 5, 10, or even 100 or more times) between the elevated pressure and the initial pressure. Such cycling can be carried out at the initial temperature, at a low temperature (e.g., subzero temperatures such as between xe2x88x9240xc2x0 C. and 0xc2x0 C., or between xe2x88x9220xc2x0 C. and xe2x88x925xc2x0 C., or while the material is being cooled to a low temperature. In some cases, a sample at low temperature can be in the solid (i.e., frozen) state at the initial pressure, but in the liquid (i.e., molten, or thawed) state at the elevated pressure. In such cases, pressure cycling causes concomitant freeze-thaw cycling. The temporal pattern of pulsation can, optionally, be altered. During each cycle, the pressure is alternately raised and then lowered. The ratio of the time at high pressure to the time at low pressure is termed as the xe2x80x9cpulsation pattern ratio.xe2x80x9d A pulsation pattern ratio greater than 1:1 (e.g., 2:1 or more) can give optimal inactivation of contaminants in most cases, whereas a pulsation ratio less than 1:1 can give greater retention of properly folded, sensitive proteins.
The material being sterilized can be, for example, a biological sample, blood plasma, serum, living tissue, plant, animal, and human tissue, sputum, urine, feces, water, ascites, medical or military equipment, a foodstuff, a pharmaceutical preparation, or a vaccine. The material being sterilized can be initially contaminated with, for example, one or more of a bacterium, a virus, a fungus, a protist, a nucleic acid, a protein, yeast, a prion, or other infectious agent.
Any of the new methods described above can also be used to produce vaccines against specific pathogens. For example, a suspension of pathogenic cells can be obtained, sterilized by one of the new methods (e.g., the method that involves pressure cycling, and potentially freeze/thaw cycling, at a subzero temperature), and combined with an adjuvant to produce a vaccine. If there are toxins present in the suspension, these can removed (e.g., after the sterilization step).
The new methods can be carried out in a pressurization vessel. The pressurization vessel can, for example, contain a gas (e.g., air or an inert gas such as nitrogen). The gas can be involved, for example, in a cavitation process. Cavitation is pressurization in the presence of a gas, followed by a rapid depressurization, resulting in the explosion of cells as microscopic gas bubbles form. This method of cell disruption can also be termed explosive decompression.
In some cases, it can be useful to include a phase-change catalyst (e.g., glass particles) in conjunction with the material to be sterilized. The catalyst can subsequently be removed by centrifugation or filtration, if necessary. The phase change catalyst can be, for example, an endogenous component of the material to be sterilized or can be added to the material.
Materials sterilized by any of the above methods are also considered to be an aspect of the invention.
In another embodiment, the invention features an apparatus for sterilizing a material. The apparatus includes a pressurization vessel adapted to transmit an external pressure to a material within itself. The vessel needs to be capable of withstanding an elevated pressure (e.g., pressures encountered in the practice of any of the new methods described above), must be capable of fitting in a pressure cycling apparatus (e.g., such as those described in PCT US97/03232), and may include a valve that allows aseptic recovery of the sterilized material. In some cases, the apparatus can also include heating and cooling devices (e.g., a heater and a refrigerator).
In still another embodiment, the invention features a method for sterilizing a sample that includes macromolecules of interest such as proteins, nucleic acids, nutrients, drugs, lipids, steroids, carbohydrates, or members of two or more classes of such macromolecules. The method includes the step of providing the sample at an initial pressure; rapidly increasing the pressure to a pressure sufficient to inactivate pathogens; and quickly restoring the initial pressure to provide a sterilized sample and to avoid substantial aggregation, denaturation, or inactivation of the biological activity of the proteins or other macromolecules.
In yet another embodiment, the invention features another method for sterilizing a sample that includes proteins. The method includes the steps of providing the sample at an initial pressure; adding one or more protein stabilizing reagents (e.g., sugars such as glucose; glycerol; a hydrophilic polymer; a cyclodextrin; a caprylate; acetyl tryptophanoate; polyethylene glycol; an anti-oxidant; or a protein specific ligand) to the sample; increasing the pressure to an enhanced pressure (e.g., about 10,000 to 70,000 psi, depending on the stability of the protein); incubating the sample for a time sufficient for sterilization to occur without substantial loss of protein function; and restoring the pressure to the initial pressure, to provide a sterilized sample.
The invention also features a method for disruption of cells or tissue or inactivation of microbes. The method includes the steps of freezing the sample; and pulsating the pressure while maintaining the sample in the solid phase, to disrupt the cells.
Another embodiment of the invention features a method for inactivating proteins in a sample. The method includes the steps of adding to the sample a reagent containing moieties that can react with amines, thiolates, carboxylates, imidazoles, or other functional groups typically found on proteins (e.g., isothiocyanates, maleamides, succinimides, sulfonyl chlorides), to form a reaction mixture; and pressurizing the reaction mixture, to inactivate the proteins. A protein-stabilizing agent can optionally be added (e.g., prior to sterilization).
In any of the above methods, the material to be sterilized can be provided in its final packaging, the packaging being able to transmit pressure without rupture. For example, the packaging can be hermetically sealed in flexible plastic. Alternatively, the packaging can be a syringe and the pressure can be transmitted via a plunger.
The invention also features a method for pressurizing an infectious sample. The method includes the steps of charging the sample into a container adapted to transmit an external pressure to the sample; submerging the container in a sterilizing chemical solution (e.g., containing an oxidizing agent); and pressurizing the sample within the container.
As used herein, the term xe2x80x9csubzero temperaturexe2x80x9d means a temperature lower than 0xc2x0 C. (e.g., xe2x88x921xc2x0 C., xe2x88x925xc2x0, xe2x88x9210xc2x0 C., xe2x88x9220xc2x0, or lower). All temperatures herein are in degrees Celsius unless otherwise stated, and are simply denoted by xe2x80x9cxc2x0 C.xe2x80x9d. Units of pressure herein are expressed in pounds per square inch (psi) or in atmospheres (atm). 1 atmosphere is about 14.7 psi, 1 bar, or 101.3 kilopascals.
A xe2x80x9ccryobaric processxe2x80x9d is a process that involves at least one pressure change carried out at a subzero temperature. In some cryobaric processes, the pressure is cycled between two pressures (e.g., about 14.7 psi to about 35,000 psi) while the temperature is either maintained at a subzero temperature or varied within a subzero temperature range.
The terms xe2x80x9csterilizexe2x80x9d, xe2x80x9cdisinfectxe2x80x9d, and xe2x80x9cdecontaminatexe2x80x9d are used interchangeably herein, unless otherwise demanded by the context. It should be noted that xe2x80x9csterilizationxe2x80x9d (killing of all organisms) may not be synonymous in certain operations with xe2x80x9cdecontaminationxe2x80x9d when the contaminant is non-living, such as a protein or prion.
The new methods provide several advantages. For example, the methods can be carried out at subzero temperatures (e.g., between about xe2x88x9240xc2x0 C. and 0xc2x0 C.). Pressure cycling carried out at subzero temperatures can advantageously induce oscillation between different phases of water within or outside the cells or vesicles of biological contaminants. The transition between the liquid and solid states can create physical stress on membranes, walls, and vesicles, thereby facilitating the intended processes. The range of subzero temperatures generally used in the new methods is easily accessed with relatively inexpensive equipment (e.g., commercial freezer devices) that is readily available in a range of shapes and sizes to fit a specific need. Similarly, the range of pressures required for the standard operation of the methods (e.g., from about 14.7 psi to about 30,000 psi) can be generated by devices as described in PCT US97/03232.
An apparatus for sterilization of a material by a cryobaric process will generally include a chamber for containing the material, the chamber being capable of operation at a selected elevated pressure: and a system for controlling, altering, or regulating the temperature and pressure within the chamber. The apparatus will also provide systems for removing a sterilized material in an aseptic manner from the chamber. Additionally, a typical sterilization apparatus for use with the new methods can include a variety of controls, regulators, and temperature, or pressure sensors. The pressurizing medium can be, for example, a water/ethylene glycol solution or other non-freezing solution or a solid such as powdered talc.
Variation of temperature can aid lysis of microbes and contaminating biological materials. Different types of cell membranes, walls, or vesicles can necessitate different conditions of temperature, pressure, cycle count, or cycle frequency to maximize the effectiveness of the sterilization processes.
The devices necessary for carrying out the new methods can be easily adapted to conform the requirements of particular applications. For example, a small, portable device can be obtained, thereby allowing sterilization or decontamination procedures to be carried out in the field (e.g., by paramedics or military medical personnel).
The new methods are very rapid. For example, the pressure can be cycled at a frequency of about 1 mHz to about 10 Hz, allowing the entire sterilization process to be completed within five minutes or less. Heat generated by cycling frequencies greater than 10 Hz can be destructive to fragile proteins. Nonetheless, frequencies higher than 10 Hz can also be used, when the heat generated by the cycling process would not be expected to be deleterious to the specific sample, or adequate measures are taken to remove heat. Shorter cycling times have been found to provide better retention of protein activity in some cases, without compromising sterilization effectiveness. As described, for example, in co-pending application U.S. Ser. No. 09/636,149, filed Aug. 10, 2000, and incorporated herein by reference in its entirety, in experiments relating to inactivation of MS2 with retention of fVIII activity desired, cycles that included pulses of high pressure lasting 10-15 seconds provided greater than 3-log inactivation of MS2 with greater than 50% of fVIII activity retained. Shorter pulses (e.g., 1 seconds vs. 10 seconds; 10 seconds vs. 60 seconds) also afforded greater retention of LDH activity.
The new methods allow pathogenic organisms in a sample to be neutralized without concomitant aggregation, denaturation, or inactivation of proteins or other macromolecules. The new methods can avoid denaturation, which often occurs upon sterilization of biological samples using other methods.
Rapid and economical sterilization is achievable with a minimum of macromolecule aggregation, inactivation, destruction, or denaturation. The new methods can thus be advantageously used for the production of highly active vaccines. These vaccines can be superior to vaccines produced at higher temperatures, since high temperatures can cause disruption of both covalent and noncovalent bonds in macromolecules of interest such as proteins, nucleic acids, nutrients, drugs, lipids, steroids, or carbohydrates, and can lead to a greater degree of irreversible denaturation or inactivation than the methods claimed here.
Other advantages of the new methods include the avoidance or reduction of the need for addition of chemical additives to blood fractions; scalability of the process from single units to large, pooled samples or to continuous, on-line processes; and elimination of side effects of thermal inactivation processes on protein components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The invention provides a method by which a material can be sterilized or decontaminated by high pressure in the range of about 5,000 psi to about 120,000 psi (e.g., 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000 psi, or intermediate ranges). The material is adjusted, either before or after pressurization, to a particular temperature which is both compatible with preserving the desirable properties of the material, and which also allows destruction of the contaminants.
Although the temperature, pressure, number and duration of cycles, and relative timing of pressure and temperature changes can vary, the new methods are in general carried out according to the following procedure: A material is provided at initial pressure (e.g., atmospheric pressure, 14.7 psi) and temperature (e.g., ambient temperature or higher or lower temperatures such as xe2x88x9240xc2x0 C., xe2x88x9235xc2x0 C., xe2x88x9230xc2x0 C., xe2x88x9225xc2x0 C., 20xc2x0 C., xe2x88x9215xc2x0 C., xe2x88x9210xc2x0 C., xe2x88x925xc2x0 C., 0xc2x0 C., 4xc2x0 C., 5xc2x0 C., 10xc2x0 C., 15xc2x0 C., 20xc2x0 C., 25xc2x0 C., 30xc2x0 C., 35xc2x0 C., 37xc2x0 C., 40xc2x0 C., 45xc2x0 C., 50xc2x0 C., 55xc2x0 C., 60xc2x0 C., 65xc2x0 C., 70xc2x0 C., 75xc2x0 C.,80xc2x0 C., 85xc2x0 C., 90xc2x0 C., 95xc2x0 C., or intermediate ranges). The material is then pressurized to an elevated pressure (e.g., in the range of about 5,000 psi to about 120,000 psi, e.g., 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000 psi, or intermediate ranges). The temperature can optionally be decreased (e.g., to a subzero temperature such as xe2x88x9240xc2x0 C. to 0xc2x0 C., preferably from about xe2x88x9220xc2x0 C. to about xe2x88x925xc2x0 C.) or increased (e.g., to 37xc2x0 C., 50xc2x0 C., 60xc2x0 C., 70xc2x0 C., 80xc2x0 C., 90xc2x0 C., or 95xc2x0 C.). In some cases, a temperature of 100xc2x0 C. or higher can be useful (e.g., when the macromolecules of interest are thermostable or when the preservation of macromolecule activity is not sought). The pressure can then be cycled repeatedly between the elevated pressure and ambient pressure. The sample can be produced in a frozen state after the final depressurization, or can be warmed to 0xc2x0 C. or higher before depressurization to produce a nonfrozen, sterilized product.
Other operational steps can be combined with these unit operations. For example, although a sample can be suspended or dissolved in a solvent or solution and the suspension or solution can be pressurized directly, induction of cavitation in a liquid material can be useful (e.g., for sterilizing tissue samples). In this process, the sample is pressurized along with a gaseous headspace followed by rapid release of pressure. This process can produce microscopic gas bubbles in the sample.
For most applications, an air headspace would suffice for cavitation. If fragile proteins are to be isolated, however, it may be preferable to use an inert gas such as nitrogen. Again, pulsing can be carried out at a frequency of about 1 mHz to about 10 Hz. More rapid pulsing (e.g., in the ultrasonic range) can be used if the heat generated does not damage critical components such as proteins in the sample.
The extent of cavitation necessary for sterilization depends on the type of biological contaminant and the goal of the sterilization process. For example, microbes that include a cell wall (e.g., yeast) can require more rigorous cavitation. If sterilization is intended to destroy microbial cells without significantly decreasing the biological activity of the macromolecules (e.g., proteins; nucleic acids) within those cells, then it can be preferable to use an inert gas for cavitation. The ratio of liquid to gas in most cavitation methods used with the new sterilization methods will be about 1:1 to about 1:10, although ratios outside of this range may also be used. The process differs from known sterilization methods in that either much higher pressures or else cycles of high and low pressures are used. Diffusion of gases is more rapid, and microbe destruction is greater.
The above processes can also be used in conjunction with methods in which a known static pressure and/or a particular pressure is maintained for a given time. In some cases, the pressure may be maintained at a greatly elevated level for an amount of time sufficient for microbe inactivation but insufficient for irreversible protein denaturation.
If a phase change is involved in any sterilization process, a catalyst may be added to accelerate the change. For example, finely-divided glass or other materials can serve as nucleation sites for freezing of a sample or material, or as a site for inducing nucleation of bubbles.
Although nearly any biological or non-biological material can be sterilized, decontaminated, or disinfected by the new methods, biological materials containing fragile proteins in relatively low quantities (e.g., ng, pg, or even fg range) or high concentrations can present a special problem, especially if the integrity of the proteins is to be maintained throughout the sterilization process.
Applications of the new methods include the sterilization of blood plasma from donors (e.g., for use in transfusions), sterilization of water, sterilization of foodstuffs (e.g., jams, jellies, meat-, fruit-, or vegetable-derived products, fruit juice, apple cider, milk), sterilization of ascites, decontamination of medical equipment (e.g., surgical and dental instruments such as scalpels, blades, and drills), neutralizing medical samples (blood, urine, fecal, sputum, hair, biopsy, or other tissue samples) or military equipment (e.g., instrumentation and integrated circuits) to destroy infectious agents, production of pharmaceuticals (e.g., generation of antisense drugs from biological sources), and disinfection of dry goods (e.g., clothing, bedding, and linens). The new methods can be used to arrest the growth of incubated materials or to sterilize such materials. The new methods can also be used to ensure sterility of cosmetics, pharmaceutics, and industrial products.
Examples of microbes that can be inactivated by the new methods include both hydrophilic and lipophilic viruses; nearly any bacteria, including, for example, Staphylococci, Micrococci, Pyogenic streptococci, diphtheroids (e.g., lipophilic, non-lipophilic, anaerobic diphtheroids such as Propiobacterium), gram-negative enteric bacilli (e.g., Escherichia, Enterobacter, Klebsiella, Proteus, Serratia), Neisseria, aerobic spore formers, mycobacteria; fungi, including, for example, yeast, Pityrosporum ovale, Pityrosporum orbiculare, Candida albicans, Candida parapsilosis, Torulopsis glabarata, and filamentous dermatophytic species; protists and lower multicellular organisms, including protozoan parasites; and helminth parasites; malaria-inducing organisms; prions; giardia; and other infectious agents.
Plasma pools often contain hepatitis C virus (HCV). Procedures for producing blood products can thus benefit from a process that inactivates HCV and other viruses. Human parvovirus B19 (B19), is another common contaminant of plasma. Hepatitis A virus (HAV) contaminants are less common, but still troublesome. Both B19 and HAV are small (about 15-30 nm), do not possess an outer envelope composed of lipids (non-enveloped), are resistant to heat and chemical treatment, and are difficult to remove by nanofiltration. Enveloped viruses (e.g., HIV, HBV, HCV) are also potential targets of the new methods. Currently uncharacterized viruses, such as some newly-recognized forms of hepatitis virus and transfusion transmitted virus (TTV) can also be vulnerable to the new methods. In addition, prion-based infectious agents such as transmissible spongiform encephalopathies are difficult to screen and to inactivate. However, because the methods of the invention can, under suitable conditions, induce protein unfolding, it may be possible to inactivate such agents by the methods of the invention.
Due to the possibility that disrupted virus particles can re-assemble after pressure treatment, it can be desirable to irreversibly degrade the nucleic acids contained in the virus. Moderately high pressures (e.g. 20,000 psi to 60,000 psi) can disrupt complexes of nucleases and their endogenous inhibitors. Additionally, moderate pressure can accelerate the activity of uninhibited enzymes. The process may be enhanced by the addition of nucleases. It is desirable in some cases to add a magnesium independent nuclease, as in the treatment of citrated plasma.
Alternatively, much higher pressures (e.g. 50,000 psi to 150,000 psi) can be used for sterilization of materials that are pressure-stable, such as small molecule pharmaceuticals or thermostable proteins. A pressure-cycling freeze-thaw sterilization method (e.g., a method that takes advantage of the cyclic formation of high pressure ice such as ice III, ice IV, ice V or ice VI) may also be used.
When the biological contaminants are relatively pressure stable and the sample contains labile proteins that need to be retained, a variation of this method can be used. In this variation, the pressure is increased rapidly (e.g., in less than 5 seconds, or less than 1 second) to a very high maximum pressure (e.g., 150,000 psi), and held at high pressure only briefly (e.g., less than 5 seconds). The pressure is then rapidly released (e.g., in less than 5 seconds, or less than 1 second). The inactivation of pathogens such as viruses can proceed at a much greater rate than the irreversible aggregation of protein molecules, especially at conditions of high pressure and low temperature that increase the solution""s viscosity. Under certain conditions of high pressure and low temperature (e.g., 110,000 psi and xe2x88x9210xc2x0 C.), high-pressure ice (i.e. ice V or ice VI) can form. Proteins that are trapped in the lattice structure of the high-pressure ice are less likely to aggregate. The high-pressure ice takes a finite amount of time to melt, this time being sufficient for the proteins in the sample to refold while trapped in the solid phase.
Pressure has also been shown to increase the activity of numerous enzymes. For example, RNase activity is accelerated by elevation of hydrostatic pressure. This effect can be exploited in conjunction with the new methods for the inactivation of viruses. RNA viruses are readily degraded following high-pressure treatment.
Experiments with pressure-treated natural urokinase indicated that amydolytic activity was highly retained (greater that 80% activity) at all temperatures between xe2x88x9240xc2x0 C. and 30xc2x0 C., with maximum activity at xe2x88x9240xc2x0 C. Recombinant urokinase was stable throughout the range of xe2x88x9240xc2x0 C. to 60xc2x0 C., with maximum activity at 2xc2x0 C.
The new methods can be used to improve the safety of blood transfusions. Plasma protein products are needed, for example, by hemophiliacs, cancer patients, and kidney dialysis patients. However, viruses and other pathogens frequently present in blood products can present a risk for patients in need of those products. Even using new filtration techniques that eliminate many cells, certain bacteria and viruses can remain in the products.
Ordinarily, blood plasma is isolated by obtaining a blood sample, centrifuging the sample in a plasma separation tube, and decanting the plasma from the precipitate in the tube. Although this method frees the plasma from the bulk of the cells, some cells inevitably remain in the plasma. If the remaining cells include, for example, bacteria or viruses, diseases can be spread by transfusion. The new methods can be carried out on the plasma obtained from the above decanting method. The contaminants that remain in the plasma can be inactivated by the new methods.
The new methods can also be used to sterilize industrial products. For example, bovine serum is often used in molecular biology laboratories for cell cultures. Microbial contamination of the source stock material from the supplier occurs infrequently; when it does happen, however, the economic costs and time delays can often be significant. Current methods for sterilization of fetal calf serum (e.g., heat or filtration) can inactivate functionally important proteins (e.g., growth hormones) and also cause variability from lot to lot. Moreover, even if the source stock material is initially sterile, it can become inadvertently contaminated upon opening in the laboratory. The new methods can be used in either a production process (e.g., batch or continuous) or used in individual laboratories for pretreatment of serum or other media prior to initiation of an experiment.
The techniques of low-temperature pressure perturbation and ultra-high pressure cavitation described above can be used advantageously in the production of vaccines. Vaccines are typically prepared by subjecting a solution of cultured pathogens to an inactivating treatment (e.g., heating, or addition and removal of chemical denaturants).
A successful vaccine preparation method should ideally result in a high degree of pathogen inactivation, but should allow the solution of pathogen to retain its ability to stimulate a protective immune response in the patient. Cryobaric procedures are well suited to meet the criteria needed for successful vaccine production: since cold, pressure-denatured proteins retain a more native-like structure than do heated or chemically denatured proteins, pressure inactivated pathogens can thus be more immunogenic. Pressure-denatured proteins are also less likely to aggregate, thereby providing higher yields of vaccine. The pressure-inactivation methods described herein can be economical on a large scale since there are generally no chemicals to add or remove and, unlike heat, pressure can be transmitted rapidly through a large sample.
In general, vaccine production by the new methods involves pulsation of pressure at sub-zero temperatures and/or ultra-high pressure cavitation treatments as described above. The specific conditions necessary for vaccine production can vary depending on the particular pathogen to be inactivated. For example, in the case of spore forming organisms, an optional pretreatment with low pressure and moderate temperature (e.g., 10,000 psi and 40xc2x0 C.) can be applied to cause the spores to germinate. The germinated spores can then be inactivated by the methods of the invention.
A method of sterilization has been described, wherein the product to be sterilized is mixed with a chemical agent that can preferentially bind to DNA or RNA and react with the nucleic acid (Radosevich, xe2x80x9cSeminars in Thrombosis and Hemostasis,xe2x80x9d Vol. 24, No. 2, pp. 157-161, 1998). In some cases, light is used to activate the chemical moiety. Disadvantages of such a method can include collateral damage to the desired molecular components of the product to be sterilized (e.g., via non-specific reaction with chemicals or irradiation, or by imperfect or slow penetration of the inactivating chemical to the interior of the pathogen). The application of elevated pressures can substantially overcome these problems by permeabilizing cells and viruses to allow entry of the inactivating chemicals. Elevated pressures can also enhance the affinity and selectivity of the molecules for the nucleic acids, thereby allowing the use of lower chemical concentrations or lower amounts of irradiation. Thus, a faster, less expensive, and more efficient method is obtained.
An apparatus for the execution of the photochemical method can include a high-pressure flow-through system such as described in PCT Appln. US96/03232, having a reaction chamber that includes at least one pressure-resistant window which can be made of a material such as quartz or sapphire, and a device for irradiation of the sample through that window. The flow of liquid is such that the entire sample passes through the irradiated area. The sample can then be collected aseptically. The sample can be introduced into the reaction chamber at high pressure or at low pressure, and then pressurized prior to irradiating.
In another embodiment of the invention, the sample is subjected to an elevated pressure for a brief time. The pressure and time are chosen to provide a high degree of pathogen inactivation, but the time is brief enough that proteins denatured by the elevated pressure conditions do not have time to aggregate into irreversible complexes to a sufficient extent before the refold into their native forms. The method can be enhanced by conditions that slow the rate of aggregation and increase the rate of protein refolding. For example, low temperatures or very high pressures can slow the rate of protein aggregation, and the addition of glucose can increase the rate of protein folding.
A variety of chemicals (e.g. iodine, ethyleneimine, ascorbic acid, thiophosphamide, congo red, paraformaldehyde) can be used to sterilize solutions containing labile proteins. Use of such chemicals can have negative effects, however, including slow inactivation, potential for protein damage, or the inability of compounds to penetrate to the interior of the pathogen. Elevated pressure can enhance the sterilization activity of these chemicals without exacerbating the negative effects.
In some cases, it may be desirable to sterilize a solution or other sample containing an unstable protein that would be irreversibly denatured at the pressure necessary for the sterilization procedures described above. In these cases, a stabilizing agent (e.g., amino acids such as amino acids, such as glycine, or specific ligands of proteins in the mixture, ligands of proteins to be recovered, or sugars such as glycerol, xylose, or glucose) can be added to the sample prior to pressurization. For example, caprylate and acetyl tryptophanoate can be added to blood plasma samples, and the plasma samples can then be subjected to the cryobaric sterilization process without excessive destabilization of specific plasma proteins. The stabilizer can then be removed by standard methods (e.g. dialysis, filtration, chromatography).
Hydrostatic or pulsating pressure can be a useful tool for sterilization, cell and virus disruption, and nuclease inactivation for samples that may potentially contain agents of infectious disease. Moreover, general safety considerations call for the prevention of infection of the persons handling the sample and the avoidance of contamination of other samples. One way to prevent such contamination is to use a sterilizing solution (e.g., 10% Clorox(copyright) bleach) or other oxidizing agent as a pressurizing medium.
For example, the sample can be placed inside an enclosed and flexible container, which can then be immersed in the chemical sterilizing solution. The solution can then be sealed inside of a second, chemically inert container (i.e., to keep it from contacting the metal parts on the inside of a pressurization chamber). An inert pressurizing medium can then be used to fill the volume between the inside of pressurization chamber and the container holding the sample and sterilizing solution. The container that holds the sterilizing solution can be, for example, a plastic bag, a screw top plastic container, a capped syringe, or a shrink-wrapping.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.